Specificity ofthe CarboxypeptidaseInhibitor from Potatoes'However, a strong association between the inhibitor and Streptomyces ... DFP,diisopropyl fluorophosphate. Sigma with the exception

Plant Physiol. (1981) 67, 754-7580032-0889/8 1/67/0754/05/$00.50/0

Specificity of the Carboxypeptidase Inhibitor from Potatoes'Received for publication August 12, 1980 and in revised form October 27, 1980

G. MICHAEL HASS', SCOTT P. AGER, DUANE LE TOURNEAU, AND JUDITH E. DERR-MAKUSDepartment of Bacteriology and Biochemistry, University of Idaho, Moscow, Idaho 83843DONALD J. MAKUS3Department ofPlant and Soil Science, University of Idaho, Moscow, Idaho 83843

ABSTRACT

Carboxypeptidases from animal, plant, fungal, and bacterial sourceswere tested for their ability to bind to the carboxypeptidase inhibitor fromRusset Burbank potatoes. Enzymes which participate in the degradationof dietary protein were partially purfied from animal species as diverse asthe cow and the limpet, and all were potently affected by the inhibitor.However, several zymogens of the enzymes in this group were tested andshown not to bind immobiized inhibitor. With the exception of an enzymefrom mast cells and a novel carboxypeptidase A-like enzyme from bovineplacenta, all animal carboxypeptidases which were not of digestive tractorigin were not affected by the inhibitor. The inhibitor had no effect on theenzymic activities of all plant and most microbial carboxypeptidases.However, a strong association between the inhibitor and Streptomycesgriseus carboxypeptidase has been noted previously and a low afflnity (K,about 10 micromolar) for a carboxypeptidase G1 from an acinetobacteriumwas found in this study.

Although proteinaceous inhibitors of the serine endopeptidasesare widely distributed in the plant and animal kingdoms (forreview see 18), only potatoes (26, 28), tomatoes (16), and round-worms (17) are known to contain specific inhibitors of the pan-creatic carboxypeptidases. In this study we have attempted toidentify which of a variety of carboxypeptidases are affected bythe inhibitor from potatoes. This information is of interest in thatit may elucidate the evolution of this enzyme class, determinewhich enzymes might be amenable to purification by affinitychromatography on immobilized inhibitor (1), and, perhaps, clar-ify the physiological role of the inhibitor.

MATERIALS AND METHODS

Materials. Carboxypeptidase isoinhibitor II (CPI4) was purifiedfrom Russet Burbank potatoes as described by Ryan et al. (28,15). CPI was coupled to CNBr-activated Sepharose 4B (PharmaciaFine Chemicals) as described previously (I). Derivatized resin hada capacity of 6 mg of bovine carboxypeptidase A bound per ml ofpacked gel. All peptide and ester substrates were purchased from

' This work was supported in part by Grant GM 22748 from theNational Institutes of Health. Publication is with the approval of theDirector of the Idaho Agricultural Experiment Station as Research PaperNo. 80512.2To whom reprint requests should be addressed.3Present address: Department of Agricultural Chemistry, Washington

State University, Pullman, Washington 99163.4Abbreviations: CPI, carboxypeptidase isoinhibitor II; CPDase, carbox-

ypeptidase; DFP, diisopropyl fluorophosphate.

Sigma with the exception of CBZ-Pro-Alas and CBZ-Leu-Phewhich were products of Vega Biochemicals. All other chemicalswere reagent grade or better and were used without furtherpurification.

Animal Enzyme Sources and Assays. Bovine CPDase A waspurchased from Sigma and bovine CPDase B, porcine CPDase A,and porcine CPDase B were prepared from trypsin-activatedextracts ofpancreatic acetone powders by affinity chromatography(1). Lungfish procarboxypeptidase B was the generous gift of Dr.Christoph de Haen, of the Department of Medicine, University ofWashington. Crude preparations of the bovine and porcine zy-mogens were aqueous extracts (4 C, I h) of pancreatic acetonepowder which had been centrifuged at 15,000 rpm for 30 min.CPDase A activity was determined spectrophotometrically usingI mM hippuryl-Phe as substrate (10). CPDase B activity wassimilarly determined using 1 mm hippuryl-Arg (32). The corre-sponding zymogens were assayed after incubation with 1% (w/w)bovine trypsin at pH 7.5 for I h at 37 C.

Third instar larvae (5 g) of the cabbage looper (Trichoplusia ni)were homogenized in 50 ml of 0.5 M NaCl, 10 mM Mes (pH 6.0)at 4 C in a Waring Blendor for three 1-min periods. The homog-enate was filtered through glass wool and then centrifuged at24,000g for 30 min at 4 C. The supernatant was assayed spectro-photometrically for CPDase activity using I mm hippuryl-DL-phenyllactate in 0.5 M NaCl, 25 mm Tris-HCI (pH 7.5) (20).A CPDase A-like enzyme was detected in extracts of bovine

placenta using hippuryl-DL-phenyllactate as substrate (20) (seeabove). The protein fraction which precipitated at 0 to 50%osaturation with ammonium sulfate was generously supplied byDr. Garth Sasser of the Department ofAnimal Science, Universityof Idaho.A preparation containing catheptic CPDase B was obtained by

fractional precipitation with ammonium sulfate of an acid extractof bovine spleen tissue (13). A suitable aliquot of enzyme wasincubated at 37 C in 1 ml of sodium acetate, 10 mm 2-mercapto-ethanol (pH 4.0) containing 10 mm hippuryl-Arg as substrate. Atvarious times 0.1 ml aliquots of assay solution were mixed with0.5 ml of 5% trichloroacetic acid, centrifuged, and free argininewas measured in the supernatant by automatic amino acid analysis(13).

Solubilized and partially purified swine kidney CPDase P(through the "toluene-trypsin step") was prepared from 100 g offresh tissue as previously described (7). Enzyme assays wereperformed at 40 C using 3.9 mm CBZ-Pro-Ala in 0. 1 M NaCl, 25mm sodium barbiturate, 25 mm sodium acetate, I mm MnCl2 (pH7.75) (7).CPDase N was partially purified from human, porcine, and

bovine serum by fractional precipitation with ammonium sulfate(8). The enzyme assay was identical to that used for the pancreatic

5 In all substrates the constituent amino acids were of the L-configura-tion.

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CARBOXYPEPTIDASE INHIBITOR SPECIFICITY

CPDases B (see above).An insoluble CPDase A-like enzyme was partially purified from

chicken muscle (pectoralis major) (22). Freshly excised tissue washomogenized in a blender for 1 min in 0.5 M NaCl, 10 ims Tris-HCI (pH 7.5). After centrifugation at l0,000g for 10 min, thesupernatant was discarded, and the pellet was suspended in 10volumes of the homogenization buffer and centrifuged as above.The procedure was repeated three times to yield the pellet usedfor enzymic assay. CPDase activity was measured by incubationof the suspended precipitate at 37 C in 0.5 M NaCl, 10 mi sodiumphosphate (pH 7.0). The rate of autolytic release of phenylalaninewas monitored by automatic amino acid analysis after precipita-tion with 10%1o trichloroacetic acid.

Angiotensin-converting enzyme (kininase II) was partially pu-rified by homogenizing 30 g of rabbit lungs (Frozen Type III fromPel Freeze) in 0.25 M sucrose, 30 mm KCI, 5 mM MgCl2, 20 mMHepes (pH 7.8) at 4 C for 1 min (6). The homogenate wascentrifuged at 18,000 rpm for 30 min at 2 C and the supernatantdiscarded. After two washes the precipitate was resuspended inbuffer and a 25-,ul aliquot was added to 0.45 ml of 2 mm hippuryl-His-Leu in 0.3 M NaCl, 0.1 M sodium phosphate (pH 8.3) (6).Solutions were incubated at 37 C for various time intervals andthe reactions were stopped by the addition of 0.5-ml aliquots of10%Yo trichloroacetic acid. Substrate hydrolysis was estimated byreaction with fluorescamine (30).

Microbial Carboxypeptidase Sources and Assays. CPDase G1from an as yet unidentified acinetobacterium was the generousgift of Drs. T. Buckman and A. Aszalos of the Frederick CancerResearch Center. Enzymatic activity was measured spectropho-tometrically using leucovorin as substrate in 0.1 mm ZnCl2, 50 mMTris-HCl (pH 7.3) at 37 C as described earlier (21). Ki values wereestimated using substrate concentrations of 10 to 100 riM.CPDase G1 from Flavobacterium sp. was generously provided

by Dr. A. Albrecht of the Memorial Sloan-Kettering CancerCenter (2). The enzyme was assayed spectrophotometrically at25 C using 60 tiM folic acid in 0.1 mim ZnCl2, 50 mm Tris-HCl (pH7.3) (21).The plant pathogenic fungi, Phymatotrichum omnivorum

(ATCC 22316) and Sclerotinia sclerotiorum (19), were grown inpotato dextrose broth at 24 C, and culture filtrates were collectedafter a heavy mycelial mat had formed. In the case of P. omnivo-rum, the filtrate was collected and dialyzed against 1 M NaCl, 25mM Hepes (pH 7.5) at 4 C. The enzyme was assayed at 37 C with20 mm hippuryl-Phe in dialysis buffer (24). The release of phen-ylalanine as a function of time was monitored by reaction withninhydrin (23). For S. sclerotiorum the culture filtrate was obtainedand 50-,ul aliquots were incubated with 0.45 ml of CBZ-Leu-Tyrin 50 mM Hepes, 50 mm sodium acetate (pH 6.5). After incubationfor 18 h at 37 C, the reactions were stopped by the addition of 0.5ml of 10%7o trichloroacetic acid. The precipitate was removed bycentrifugation and free tyrosine was determined by reaction withfluorescamine (30).

Plant Enzyme Sources and Assays. CPDase C was partiallypurified from the flavedo of the lemon through the 0 to 75%ammonium sulfate fractionation step (34). The precipitate wasredissolved and dialyzed exhaustively against distilled H20 at 4 C.Aliquots of enzyme solution were mixed with equal volumes of 2mM CBZ-Leu-Phe in 0.1 M sodium citrate (pH 5.3), and assayswere conducted at 37 C for up to 24 h. Aliquots of 0.14 ml werechromatographed on Whatman 3 MM filter paper using l-bu-tanol:acetic acid:H20 (4:1:5) as solvent and the chromatogramswere stained with cadmium-ninhydrin reagent (3). The zonescorresponding to phenylalanine were eluted with methanol andthe absorbances of eluates were measured at 504 nm. Enzymeblanks were prepared, as were assays on enzyme preparationswhich had been preincubated for 15 min with 25 mm DFP.CPDase was extracted from mung bean (Phaseolus aureus)

cotyledons in cold 2 mm 2-mercaptoethanol, 25 mm citrate-phos-phate (pH 5.0) (5). After exhaustive dialysis at 4 C against theextraction buffer, assays were performed using 2 mm CBZ-Phe-Ala in 2 mm 2-mercaptoethanol, 0.5 mm EDTA, 25 mm citrate-phosphate (pH 5.0). Alanine was detected after paper chromatog-raphy as described for the lemon peel enzyme.Tomato fruit (Lycopersicon esculentum) was homogenized at

4 C for I min and the homogenate was centrifuged at 9,000 rpmfor 30 min at 4 C. The clear extract was adjusted to 0.5 M NaCl,50 mm Tris-HCl (pH 7.5) and chromatographed on a column ofCPDase A-Sepharose 4B to remove the endogenous CPDaseinhibitor (15, 16). The breakthrough fraction was adjusted to 70%saturation with solid ammonium sulfate and then centrifuged at9,000 rpm for 30 min. A portion of the precipitate was dissolvedin 50 mm sodium acetate (pH 4.4) and the rest was dissolved in 50mM Hepes (pH 7.5). After dialysis against the respective buffers,assays were conducted at 37 C using CBZ-Gly-Phe at both pHvalues. Release of phenylalanine was monitored as described forthe lemon peel enzyme.Wheat (Triticum aestivum) was germinated and the shoots and

roots were extracted in cold 0.2 M sodium acetate (pH 4.4) (25).The protein fraction precipitating at 35 to 70% saturation ofammonium sulfate was dialyzed exhaustively against extractionbuffer and assayed at 37 C with 2 mm CBZ-Phe-Ala in 0.5 mMEDTA, 50 mm sodium acetate (pH 5.7) (25). An aliquot (50 1I)was added to 150 t,l of 5% HC104, and after 30 min in an ice baththe precipitate was removed by centrifugation. An aliquot (40 tl)of the supernatant was examined for free alanine by reaction witho-phthaldialdehyde (27).

Pine seedlings (Pinus sylvestris), the generous gift of Dr. FrankPitkin of the Dept. of Forestry, University of Idaho, were homog-enized in cold 50 mm sodium phosphate (pH 6.5) containing I mnmDTT (29). Assays were conducted at 30 C using 2 mm CBZ-Phe-Ala as substrate in 0.1 M sodium acetate, I mm EDTA (pH 4.2)(29). The release of alanine was monitored as described for thelemon peel enzyme (see above).

Binding of CPDases to CPI. The affinity of the CPDases forCPI was assessed by two procedures. Most commonly CPI wasincluded in assay solutions at a concentration of approximately 10/LM, and the time course of the hydrolysis of substrate was com-pared with assays performed in the absence of the inhibitor.

In some cases the binding of enzyme to immobilized CPI wasmonitored. This procedure was used for the zymogens, the pla-centa CPDase and the CPDases G1. Enzyme or zymogen wasdissolved in 0.5 M NaCl, 50 mm Tris-HCl (pH 7.5) and applied toCPI-Sepharose. The amount of enzyme activity eluting in thebreakthrough fractions was compared with that applied to thecolumn.

RESULTS

The association between CPDases and CPI was assessed by oneor both of the following procedures. The most common methodwas to monitor the decrease in enzymic activity observed in thepresence of inhibitor (about 10 tiM). Although this approach wouldreadily detect tight enzyme-inhibitor associations, either weakbinding (Ki > 10 tsM) and/or high substrate/Km ratios in the assaysolutions could produce a false negative result. The other proce-dure was to observe the binding of enzyme or zymogen to im-mobilized CPI. Since the effective CPI concentration on the resinwas typically 20-fold greater than that in inhibition studies andsince no substrate was present to compete with inhibitor, muchweaker enzyme-inhibitor binding could be detected by thismethod. However, the possibility of some nonspecific binding ofenzyme must be considered in evaluating data of this type.The information on the inhibition of various CPDases as pre-

sented in Tables I to III includes both new data and some materialwhich has been published previously. The latter data are desig-

Plant Physiol. Vol. 67, 1981 755



Plant Physiol. Vol. 67, 1981

nated in the tables by their appropriate references.Animal CPDases. CPI, originally detected by its ability to

inhibit porcine CPDase B (26), potently affects CPDases from thedigestive tracts of animal species as diverse as the cow and thelimpet (Table I). Ki values of 5 nM, 50 nm, and 2 nM have beenreported for bovine CPDase A (28), porcine CPDase B (28), andthe limpet CPDase (14), respectively. Ki values for the inhibitionby CPI of the other enzymes listed in Table I have not beendetermined; however, greater than 95% inhibition is observed forthese enzymes at CPI concentrations of approximately 10 ,UM.Thus, Ki values of 0.1 UM or lower would be expected in eachcase. No examples have been found of animal CPDases whichfunction to degrade dietary protein and are not inhibited by CPI.The CPDase A-like enzyme activity observed in homogenates ofwhole insects (cabbage looper) presumably is from the digestivetract; however, this has not been clearly established.

Digestive CPDases are often synthesized as zymogens whichlack, or possess greatly reduced, enzymic activity. Porcine andbovine procarboxypeptidases A and B and the procarboxypepti-dase B from the lungfish were not retarded when applied to CPI-Sepharose 4B. Since the resin had an effective capacity of 6 mgbovine CPDase A bound per ml of packed gel (i.e. about 0.17mM), any association between these zymogens and the inhibitorwould have to be quite weak to go undetected in this study. Inaddition, the intrinsic enzymic activity of bovine procarboxypep-tidase A was not affected by CPI (10 ,(M), providing furtherevidence that the inhibitor binding site is either blocked or dis-torted in the zymogen (28).

Several animal CPDases which do not participate in the deg-radation of dietary protein have also been tested for susceptibilityto CPI (Table II). Of these, only CPDase from rat peritoneal mastcells (9) and from bovine placenta appear to be affected by CPI.Unfortunately, no data on the strength of binding of CPI to themast cell protease are available; however, preliminary data on theplacental enzyme (G. M. Hass, unpublished observations) suggestthat it has a Ki value of approximately 0.1 LM for CPI. Therelationship between these two enzymes and bovine CPDase Amerits further examination.

Microbial CPDases. Several bacterial and fungal CPDases weretested for their susceptibility to CPI (Table III). Of these studies

Table II. Binding of CPI to Intracellular and RegulatoryCarboxypeptidases

Procedure

Inhibi- Bindingtion of to CPI-Enzymic Sepha-Activity rose

Mast cell CDPase (rat) + (9)8 + (9)Placenta CPDase (bovine) + +Cathepsin A (bovine spleen) - (28) NDbCatheptic CPDase B (bovine spleen) - NDCPDase P (bovine kidney) - NDCPDase N (human, bovine, porcine serum) -

CPDase (chicken muscle) - NDKininase II - ND8 Data which include a reference number are taken from the literature.b Not determined.

Table III. Binding of CPI to Microbial Carboxypeptidases

Procedure

Enzyme Inhibition of BindingEnzymic to CPI-Activity Sepharose

CPDase GI (P. stutzeri) + (28)8 NDbCPDase G, (Acinetobacter sp.) + +CPDase GI (Flavobacterium sp.) -S. griseus CPDase + (11) +Yeast protease a - (28) NDYeast protease Y - (28) NDP. omnivorum CPDase - NDS. sclerotiorum CPDase - ND

8 Data which include a reference number are taken from the literature.b Not determined.

SPERMATOPHYTA

Table I. Binding of CPI to Digestive Tract Carboxypeptidases and toTheir Zymogens

Procedure

Enzyme Inhibition of Binding toEnzymic CPI-Sepha-Activity rose

Bovine CPDase A + (28)8 + (1)Bovine CPDase B + +(1)Porcine CPDase A + +(I)Porcine CPDase B + (28) + (1)Shrimp CPDase A + NDbShrimp CPDase B + NDLungfish CPDase B + NDCrayfish CPDase + (35) + (35)Cabbage Looper CPDase + NDLimpet CPDase + (14) +Bovine Pro CPDase A - (28) -

Bovine Pro CPDase B NAC -

Porcine Pro CPDase A NA -

Porcine Pro CPDase B NA -

Lungfish Pro CPDase B NA -

8 Data which include a reference number are taken from the literature.b Not determined.c Not applicable.

GYMNOSPERMAE(Scotch pine)

MONOCOT(whe

ANGIOSPERMAE

YLEDONAE DICOTYLEDONAE-at)

r Z~~~~~~~~~~~~~~~POLYPETALAE

(lemon)GAMOPETALAE

(mung bean)(tomato)

FIG. 1. The plant CPDases that we studied were not inhibited by CPI.CPDases from each plant indicated were assayed in the presence andabsence of 10 tiM CPI using appropriate peptide substrates (see "Materialsand Methods" for details). The classification scheme is that of Bailey (4).

the data of Gage-White et al. (1 1) on the inhibition of the CPDasefrom S. griseus (i.e. Pronase CPDase) are, perhaps, the mostinteresting. The binding (Ki about 50 nM) of CPI to this enzymeis only slightly weaker than the CPI-CPDase A association.Two of the three CPDases G which were tested (Table III)

bound CPI, but the enzyme-inhibitor associations were quite weak(Ki > 1 ,UM). The corresponding enzyme from Flavobacterium sp.(2) was apparently not affected by CPI. Enzymes of this type arefolate antagonists of interest in cancer chemotherapy and metho-trexate rescue therapy.The remaining microbial CPDases were not affected by CPI. It

was most surprising that the enzyme from P. omnivorum was notinhibited by CPI in view of several similarities which have beennoted between this enzyme and bovine CPDase A (24).

HASS ET AL.756



CARBOXYPEPTIDASE INHIBITOR SPECIFICITY

Plant CPDases. CPDases have been identified in a wide varietyof plant species. Assays of representative enzymes from eachmajor subdivision, class, and subclass (4) (Fig. 1) in the presenceof CPI (10 saM) revealed no inhibition of activity. In contrast, DFP,a potent and irreversible inhibitor of enzymes of this type, de-creased the observed enzymic activity between 80 and 100%1o.

Since tomato fruit contains a CPDase inhibitor which is similarto CPI (16), the CPDase activity of extracts of tomato was carefullystudied with respect to possible inhibition. Extracts of tomato fruitwere treated with ammonium sulfate and then passed through acolumn of CPDase A-Sepharose 4B prior to being assayed. Thelatter step was included to remove the endogenous inhibitor whichmight mask the activity of an inhibitor-sensitive CPDase. Assayswere conducted at a pH value (pH 4.4) similar to pH optima oftypical plant CPDases and under conditions similar to those usedto monitor bovine CPDase A. As with the other plant CPDases,inhibition was complete in DFP-treated preparations, and CPIwas not inhibitory.

DISCUSSION

This investigation of the specificity of CPI from potato wasundertaken for several reasons. First, it was hoped that the inhib-itory spectrum might clarify the physiological role of this polypep-tide. It has been suggested that the proteinase inhibitors frompotatoes serve a protective function by neutralizing the effects ofthe digestive enzymes of invading pests or microorganisms (12).The potent effect of CPI on all digestive tract CPDases whichwere examined (Table I) would support this hypothesis. In thisrespect the potent inhibition of the CPDase A-like enzyme fromthe cabbage looper is particularly relevant. However, the CPDasesfrom the fungi S. sclerotiorum and P. omnivorum, species whichmight be considered models of infecting microorganisms, were notinhibited by CPI. Clearly, CPDases from several major potatopathogens should be examined as well. The possibility that theprimary physiological role of CPI is to regulate the activity of anendogenous CPDase was explored by testing several plantCPDases for susceptibility (Fig. 1). In this regard, the CPDaseactivity in tomato fruit was examined with particular care, becauseof the close phylogenetic relationship between the potato andtomato. Even though none of these enzymes was affected, it ispremature to rule out a regulatory role for CPI.The second reason for this study was to identify CPDases whose

purification would be greatly facilitated by using immobilized CPI(1). A primary interest in this regard involved enzymes of theCPDase G-type, since they are under investigation in cancerchemotherapy. Although two of the three such enzymes testedwere inhibited by CPI, their association constants for inhibitorwere too low to render immobilized CPI of value in their purifi-cation. Immobilized CPI has been utilized to prepare mast cellCPDase (9), and crayfish CPDase (35), as well as the bovine andporcine carboxypeptidases A and B (1). In addition, preliminarydata on the bovine placenta CPDase A-like enzyme indicate thatimmobilized CPI effects a purification of greater than 600-foldwith a yield of over 60%o (G. M. Hass, unpublished observations).

Third, studies which indicate that the zymogens of the pan-creatic CPDases do not bind to CPI suggest that the activationpeptide region may block or distort the normal enzyme-inhibitorcontact zone. The failure of bovine procarboxypeptidase A to bindto CPI is particularly intriguing, since this zymogen has a signifi-cant intrinsic catalytic activity (33). In fact, Uren and Neurathhave shown that at low pH the bovine zymogen is more activethan the enzyme toward certain substrates (31). It is tempting tospeculate that the activation peptide of the zymogen and CPI bindto identical or overlapping sites of the enzyme.

Finally, that a given CPDase is inhibited by CPI may be usedas evidence that this enzyme and the pancreatic CPDases haveevolved from a common protein. In this respect it is interesting to

note that all CPDases which are inhibited by CPI are metalloen-zymes. In contrast, none of the members of the other principalCPDase family, the serine CPDases, is affected by the inhibitor.Of course, that two enzymes are inhibited by CPI does not provetheir common ancestry because of the possibility of a convergentevolutionary mechanism in which similar catalytic apparatusesand CPI-binding sites were generated independently. Just asinhibition by CPI does not constitute definitive proof that a givenenzyme and the pancreatic carboxypeptidases are evolutionaryrelatives, neither does the failure of an enzyme to bind CPIexclude it from this family. Clearly, a relatively small change inthe amino acid sequence in the CPI-binding site could preventbinding. The distortion or blocking of the CPI-binding site in thezymogens could be given as an example of such a situation. Inspite of the deficiencies inherent in using CPI binding as a familymarker, this may serve as an easy test to suggest evolutionaryrelationships and to support other available evidence. The inhi-bition of a given CPDase (e.g. placenta CPDase) by CPI wouldsuggest that the battery ofgroup specific reagents, chelating agents,and affinity labels which are effective against the pancreaticenzymes should be carefully investigated in initial structure-func-tion studies.

Acknowledgments-The cooperation of Drs. Buckman, Aszalos, and Albrecht withthe studies involving carboxypeptidases G, is gratefully acknowledged as is the giftof pine seedlings from Dr. Frank Pitkin. The authors also wish to thank Dr. C. A.Ryan for his suggestions and Ms. Debra La Pointe for excellent technical assistance.

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758 HASS ET AL.

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carboxypeptidase: affinity chromatography, characterization and amino-ter-minal sequence. Eur J Biochem 94: 223-229



Plant Physiol. (1981) 68, 12100032-0889/81/68/1210/01/$00.50/0

CorrectionsVol. 67: 754-758, 1981 Vol. 67, Number 4, 1981

G. Michael Hass, Scott P. Ager, Duane Le Tournea, Judith E.Derr-Makus, and Donald J. Makus. Specificity of theCarboxypeptidase Inhibitor from Potatoes.

Page 754, author line, should be corrected to read: ... Duane LeTournea ...

Author IndexLe Tourneau, Diane, should be corrected to read: Le Tournea,Duane.

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Specificity ofthe CarboxypeptidaseInhibitor from Potatoes'However, a strong association between the inhibitor and Streptomyces ... DFP,diisopropyl fluorophosphate. Sigma with the exception